The mechanisms leading to the opposite effects of acute and chronic alcohol use on inflammatory cytokine production are yet to be explored. Recognition of inflammatory signals in monocytes is mediated via receptors including Toll-like receptors, TLR2, TLR4, and CD14, and result in activation of the NF-?B/Rel molecules, a central component of cellular responses to microbial stress. Our previous work revealed that acute alcohol, both in vivo and in vitro, inhibits NF-?B activation in monocytes and this inhibition is independent of IkBa degradation. In contrast, chronic alcohol use has been shown to increase inflammatory mediator production and, based on our preliminary results, augments NF-?B activation in LPS-stimulated monocytes. Thus, we hypothesize that acute and chronic alcohol treatment have opposing effects on NF-?B activation in human monocytes and this may mediate the distinct effects of acute and chronic alcohol use on inflammatory mediator production. We further postulate that acute and chronic alcohol target NF-?B activation at different levels of the NF-?B signaling pathway in monocytes. Specifically, we propose that acute, and not chronic alcohol, inhibits NF-?B activation via mechanisms independent of IkBa-degradation. We further postulate that inhibition or augmentation of NF-?B activation by alcohol depends on the complexity of signaling events mediated via the CD14, TLR4 and TLR2 receptors in monocytes. The Specific Aims of this proposal are: 1) To explore mechanisms, independent of IkBa degradation, for inhibition of NF-?B by acute alcohol including IkBe, p65 nuclear export, p65 phosphorylation, and Akt-mediated events. These pathways will be evaluated in vitro after acute alcohol treatment of human monocytes or CD14-transfected CHO cells and confirmed in monocytes after in vivo acute alcohol intake. 2) To reveal intracellular events leading to the diverse effects of in vitro acute and chronic alcohol treatment on NF-?B activation in human monocytes by examination of differential regulation of IkBa, lkB?_ and IkBe and NF-?B-related gene activation pathways by gene-array analysis. 3) To investigate the modulating effects of acute and chronic alcohol on the expression of TLR2, TLR4, and CD14 and cell activation via these receptors in monocytes, and to further dissect alcohol-induced changes in TLR2 and TLR4-mediated events in genetically engineered CHO and HEK cells. Results from these studies should delineate the signaling mechanisms underlying the opposing effects of acute and chronic alcohol use in human monocytes relevant to inflammatory cell activation in host defense and liver disease.